Method for screening and functional analysis of related genes for improving constipation by using multiple traditional Chinese medicine components

Abstract

The invention provides a method for screening and functional analysis of related genes for improving constipation by using multiple traditional Chinese medicine components. The method comprises the following steps: screening traditional Chinese medicine compound active ingredients and corresponding action targets, obtaining constipation disease targets, obtaining traditional Chinese medicine compound action targets and constipation disease intersection targets, constructing a traditional Chinese medicine compound active ingredient-action target regulation and control network, constructing a PPI protein interaction network, carrying out enrichment analysis on KEGG and GO, carrying out molecular docking, and carrying out in-vivo experimental verification. Compared with the prior art, the method has the advantage that core component groups and key targets in the compound can be identified, the research mode of'single component-single target 'of a single medicine is broken through, and the integrity of traditional Chinese medicine argumentation is embodied.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine functional analysis, and particularly relates to a method for screening and functional analysis of multiple traditional Chinese medicine components for improving constipation related genes. BACKGROUND

[0002] Constipation is a common gastrointestinal problem that seriously affects people's physical health and living standards. At present, volume laxatives, osmotic laxatives, stimulant laxatives and lubricant laxatives are commonly used at home and abroad to improve constipation symptoms, but these drugs have the problems of slow effect, easy abdominal distension and long-term use of intestinal nerve damage. Therefore, it is of great significance to develop efficient, non-toxic, low-cost and effective improving agents. Modern research shows that traditional Chinese medicine prescription medicines can effectively treat constipation symptoms due to their multi-target and multi-pathway characteristics. However, the active ingredients of traditional Chinese medicine prescription medicines are complex, and the selection and optimization of the prescription requires a large number of experiments. Therefore, before the experiment, simulating the mechanism of drug components and disease targets through network pharmacology is low-cost and efficient.

[0003] Compared with single traditional Chinese medicine, traditional Chinese medicine prescription has the characteristics of system and integrity. Network pharmacology, as a new discipline, can efficiently and scientifically simulate the connection between traditional Chinese medicine prescription components and target points through computer. Finally, the results of network pharmacology deduced by experiments can efficiently solve the problems of high cost and long experimental time. At present, there is only a computer simulation method for single traditional Chinese medicine treating constipation, and there is no computer simulation method for studying multi-pathway and multi-target traditional Chinese medicine prescription treating constipation. SUMMARY

[0004] The purpose of the present application is to solve the above problems existing in the background art, and to provide a method for predicting and simulating multiple traditional Chinese medicine components for screening and functional analysis of constipation related genes.

[0005] The technical solution adopted by the present application is: a method for screening and functional analysis of multiple traditional Chinese medicine components for improving constipation related genes, comprising the following steps:

[0006] Step 1: screening of traditional Chinese medicine prescription active ingredients and corresponding target points;

[0007] Step 2: obtaining of constipation disease target points;

[0008] Step 3: obtaining of intersection target points of traditional Chinese medicine prescription target points and constipation disease target points;

[0009] Step 4: construction of traditional Chinese medicine prescription active ingredient-target point regulation network;

[0010] Step 5: construction of PPI protein interaction network;

[0011] Step six, KEGG and GO enrichment analysis;

[0012] Step seven, verifying the effect of traditional Chinese medicine compound on improving constipation by zebra fish model.

[0013] The method for improving constipation related gene screening and function analysis of multi-flavor traditional Chinese medicine components can screen and analyze the function of the constipation related genes of the multi-flavor traditional Chinese medicine components through network pharmacology. And the effect of the traditional Chinese medicine compound on improving constipation is verified by the zebra fish model.

[0014] Further, step one specifically comprises: retrieving and obtaining the active components of the traditional Chinese medicine compound of yam, herba rhizomae, angelica sinensis, cistanche deserticola, medlar, astragalus, epimedium and clove in the TCMSP database, screening by taking oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18% as the standard, obtaining the action target points corresponding to the screened components by using the database, and finally standardizing the target gene name by using the Uniprot database.

[0015] Further, step two specifically comprises: collecting constipation disease targets in GeneCards database, OMIM database, TTD database, PharmGKB database by taking “constipation” as the keyword, and removing the repeated values.

[0016] Further, step three specifically comprises: importing the traditional Chinese medicine compound and the constipation disease targets into the jvenn platform to obtain the intersection targets and draw the wein diagram.

[0017] Further, step four specifically comprises: visualizing the active components of the traditional Chinese medicine compound and the action targets by using Cytoscape 3.7.1 software to obtain the active component-target network diagram of the traditional Chinese medicine compound.

[0018] Further, step five specifically comprises: importing the intersection targets obtained in step three into the STRING12.0 platform to construct the PPI protein interaction network diagram of the traditional Chinese medicine compound and constipation.

[0019] Further, step six specifically comprises: importing the intersection targets of the traditional Chinese medicine compound and constipation disease into the DAVID database for KEGG and GO enrichment analysis.

[0020] Further, step seven specifically comprises: verifying the effect of the traditional Chinese medicine compound on improving constipation by using the zebra fish model.

[0021] The beneficial effects of the present application are:

[0022] 1. For constipation disease, computer simulation prediction is carried out according to the systematic and holistic thinking of traditional Chinese medicine.

[0023] 2. Compared with the single-target-single-path mode of single Chinese medicine, the construction of the Chinese medicine formula component-target-disease network can directly analyze how each component in the compound forms a synergistic effect by regulating key targets, which has more practical application significance.

[0024] 3. The combination of network pharmacology and experimental verification can improve the persuasiveness of the experiment. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 A network diagram of active ingredients and action target regulation of traditional Chinese medicine compound, wherein shanyao-shanyao, xianmao- xianmao, danggui-danggui, roucongrong-roucongrong, gouqizi-gouqizi, huangqi-huangqi, yinyanghuo-yinyanghuo, dingxiang-dingxiang; the left circle is the gene target, and the right circle is the active ingredient in the traditional Chinese medicine compound;

[0026] Figure 2 A target point of constipation disease and active ingredient of traditional Chinese medicine compound;

[0027] Figure 3 A network diagram of traditional Chinese medicine compound and constipation PPI protein interaction;

[0028] Figure 4 A core target point diagram of traditional Chinese medicine compound and constipation disease target point PPI network;

[0029] Figure 5 A GO enrichment diagram of potential target points of active ingredients of traditional Chinese medicine compound;

[0030] Figure 6 A KEGG enrichment diagram of potential target points of active ingredients of traditional Chinese medicine compound;

[0031] Figure 7 A diagram of the food excretion promoting effect of the traditional Chinese medicine compound extract on zebra fish. DETAILED DESCRIPTION

[0032] The present application will be described in detail below in conjunction with specific examples and drawings. It should be understood that the examples are only used to illustrate the present application, and are not used to limit the present application, and any modification, equivalent replacement, etc. made on the basis of the present application is within the protection scope of the present application.

[0033] The experimental methods in the following examples are conventional methods, and the test materials used in the following examples are commercially available unless otherwise specified.

[0034] Example 1

[0035] The present embodiment indicates a method for screening and functional analysis of multi-flavor traditional Chinese medicine components to improve constipation-related genes, and the method steps are as follows:

[0036] (1) Screening of active components of traditional Chinese medicine compound and corresponding target points. The active components of Shanyao, Xianmao, Danggui, Roucongrong, Gouqi, Huangqi, Yinyanghe and Dingshang of traditional Chinese medicine compound were retrieved and obtained by using TCMSP database (https: / / tcmsp-e.com / tcmspsearch.php). The screening was performed by using oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18% as the standard, and the corresponding target points of the screened components were obtained by using the database. Finally, the target gene name was standardized by using Uniprot database (https: / / www.uniprot.org / ).

[0037] (2) Acquisition of constipation disease target points. With “Constipation” as the keyword, the constipation disease target points were collected in GeneCards database (https: / / www.genecards.org / ), OMIM database (https: / / www.omim.org / ), TTD database (https: / / db.idrblab.net / ttd / ), and PharmGKB database (https: / / www.pharmgkb.org / ), and the repeated values were removed.

[0038] (3) Acquisition of intersection target points of traditional Chinese medicine compound target points and constipation disease target points. The traditional Chinese medicine compound target points and constipation disease target points were introduced into jvenn platform (https: / / jvenn.toulouse.inrae.fr / app / example.html) to obtain the intersection target points and draw the Venn diagram.

[0039] (4) Construction of traditional Chinese medicine compound active component-target point network. The active components of traditional Chinese medicine compound and the target points were visualized and plotted by using Cytoscape 3.7.1 software, and the traditional Chinese medicine compound active component-target point regulation network diagram was obtained.

[0040] (5) Construction of PPI protein interaction network. The intersection target points obtained in step four were introduced into STRING12.0 platform (https: / / cn.string-db.org / cgi / input) to construct the PPI protein interaction network diagram of traditional Chinese medicine compound and constipation.

[0041] (6) KEGG and GO enrichment analysis. The intersection target points of traditional Chinese medicine compound and constipation disease were introduced into DAVID database (https: / / davidbioinformatics.nih.gov / ) for KEGG and GO enrichment analysis.

[0042] (7) Zebrafish experiment verification. The effect of traditional Chinese medicine compound on improving constipation was verified by using model organism zebrafish.

[0043] Results

[0044] ①Screening of active ingredients of traditional Chinese medicine compound and corresponding target points.

[0045] Screening was performed according to the standards of oral bioavailability (OB) ≥ 30% and drug-like property (DL) ≥ 0.18%. A total of 11 chemical ingredients of yam, 7 of caulis herba, 2 of angelica, 4 of cistanche, 45 of medlar, 20 of astragalus, 23 of epimedium and 6 of clove were obtained.

[0046] Table 1 Main active ingredients in traditional Chinese medicine compound for improving constipation

[0047]

[0048]

[0049]

[0050]

[0051] ②Visualization of active ingredients of traditional Chinese medicine compound and target points was performed by using Cytoscape 3.7.1 software, as shown in Figure 1 .

[0052] ③A total of 3,039 disease target points related to constipation were screened from OMIM, GeneCard, TTD and PharmGKB databases with “Constipation” as the subject term. Common target points of constipation disease and drugs were obtained by using jvenn platform, and a wein diagram was drawn, as shown in Figure 2 . A total of 121 potential target points for improving constipation were obtained.

[0053] ④PPI network construction. The intersection target points were imported into STRING database to obtain PPI network, as shown in Figure 3 . Then, the core target points in the top 20 were obtained by sorting the degree value by using cytoNCA plug-in of Cytoscape software, as shown in Figure 4 .

[0054] ⑤Signal pathway analysis of active target points of traditional Chinese medicine compound for improving constipation disease was performed by using Metascape data platform. The results showed that the functions of multiple protein target points were related to constipation disease. GO analysis and KEGG enrichment results are shown in Figure 5 and Figure 6The results analysis showed that the biological processes (BP) involved in the 121 potential therapeutic targets mainly included positive regulation of gene expression, response to probiotic stimulus, positive regulation of MAPK cascade, etc., the cellular components (CC) mainly included cytosol, cell surface, mitochondrion, etc., the molecular functions (MF) mainly included enzyme binding, protein binding, identical protein binding, etc., and the KEGG signal pathways mainly included cancer pathway, cytokine-cytokine receptor interaction, interleukin (IL)-17 signaling pathway, lipid and atherosclerosis, endocrine resistance, etc.

[0055] Example 2

[0056] This example verifies the effect of traditional Chinese medicine compound on improving constipation by zebrafish model.

[0057] 1. Materials and instruments

[0058] The zebrafish were bred in fish water at 28°C (water quality: 200 mg of instant sea salt was added to 1 L of reverse osmosis water, the conductivity was 450-550 μS / cm, the pH was 6.5-8.5, and the hardness was 50-100 mg / L CaCO3), which was provided by the fish breeding center of the company. The wild type AB strain zebrafish was bred by natural pair mating; sodium chloride, sodium bicarbonate, magnesium sulfate heptahydrate, calcium chloride dihydrate (National Pharmaceutical Group), Nile red (item number 19123, sigma), domperidone (Xian Yangsen), anhydrous ethanol (National Pharmaceutical Group). Traditional Chinese medicine compound extract (Jin brand Co., Ltd.).

[0059] Stemi 508 stereomicroscope, Zeiss V16 fluorescence stereomicroscope, biochemical incubator (Shanghai Boxin SPX-250B-Z type), AZ100 multifunctional continuous zoom microscope (Nikon Corporation).

[0060] 2. Experimental method

[0061] 2.1 Preparation of experimental solutions

[0062] Experimental water: deionized water or water of equivalent purity was used to prepare E3 culture solution:

[0063] Preparation method of 60*E3 culture solution: NaCl: 344 mg, KCl: 15.2 mg, CaCl2·2H2O: 58 mg, MgSO4·7H2O: 98 mg, and constant volume to 20 mL, which was diluted before use.

[0064] Preparation of Nile red: methanol was used to prepare a mother liquor with a mass concentration of 1 mg / mL, which was stored at -20°C. When used, it was diluted with E3 culture solution and stored at 4°C.

[0065] Preparation of Domperidone: Dissolve in E3 medium to make 10 mg / mL stock solution, store at 4℃.

[0066] 2.2 Preparation of fish for experiment

[0067] Remove the eggs with obvious abnormal cell division (asymmetry, vesicle) or cell membrane damage. Remove the dead and abnormal embryos every day. Under the dissecting microscope, select the 3 dpf wild type AB strain larvae without pericardial edema, without venous congestion, and with normal development (uniform size of yolk sac). Select the 5 dpf zebrafish after fertilization with normal body size, fully inflated swim bladder, normal suspended swimming, and similar body length.

[0068] 2.3 Sample preparation

[0069] Dissolve the traditional Chinese medicine compound extract in E3 medium to 5 mg / mL.

[0070] 3. MTC experiment

[0071] Determination of maximum tolerable concentration (MTC): Randomly select 6 dpf wild type AB strain zebrafish in a 6-well plate, with 30 zebrafish per well (experimental group). Dissolve domperidone in water, and set up normal control group and model control group, with a volume of 3 mL per well. After 24 h of treatment at 28℃, determine the maximum tolerable dose MTC.

[0072] 4. Evaluation of efficacy in improving constipation

[0073] Select 5 dpf zebrafish after fertilization with normal body size, fully inflated swim bladder, normal suspended swimming, and similar body length. Add Nile red (final concentration 10 ng / mL) to the container containing 5 dpf zebrafish after fertilization, and expose overnight for 16 h to make the zebrafish intestinal tract fully filled with Nile red. After Nile red exposure, wash the zebrafish with E3 medium and transfer them to a 6-well plate, with 30 zebrafish per well. Add samples to E3 medium, and set up positive control group (domperidone solution) and model control group, and set up solvent control group as blank group if necessary. The total volume of all groups is 3 mL. Mix thoroughly, wrap the 6-well plate with tin foil, and incubate in a biochemical incubator for 24 h (28℃) to the end of the experiment. Then randomly select 10 zebrafish from each experimental group and take photos under a fluorescence microscope. Analyze the images using ZEN software, and set the software analysis parameters to brightness sum to calculate the residual Nile red fluorescence signal value in the zebrafish intestine.

[0074] 5. Data processing

[0075] The images were analyzed using ZEN software, with the analysis parameter set to the sum of brightness, to calculate the residual Nile red fluorescence signal value in the zebrafish intestine. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, with P < 0.05 indicating statistical significance.

[0076] Quantitative calculations for improving constipation function:

[0077] The constipation-improving effect of the samples was calculated based on the average Nile red fluorescence signal value S in the intestines of zebrafish in each group (expressed as the rate of food excretion promotion), using the following formula:

[0078]

[0079] Experimental results:

[0080] 1. MTC Measurement

[0081] Table 1. Concentration-mortality rate of MTC experiments on compound extracts of traditional Chinese medicine.

[0082]

[0083] As shown in the table above, the maximum tolerated dose of the compound extract of traditional Chinese medicine is 2000 μg / mL.

[0084] 2. Evaluation of the effect on promoting intestinal peristalsis in zebrafish

[0085] The effect of traditional Chinese medicine compound extract on the excretion of food in zebrafish is as follows: Figure 7 As shown. From Figure 7 It can be seen that the compound extract of traditional Chinese medicine has a significant effect on promoting excretion in zebrafish, indicating that the compound extract of traditional Chinese medicine has a significant effect on improving constipation. Moreover, within the concentration range of 500-2000 μg / mL, the effect of the compound extract of traditional Chinese medicine on improving constipation is stronger as the concentration increases.

[0086] Based on the above analysis results, the method for screening and functional analysis of genes related to constipation improvement by multiple Chinese herbal ingredients provided by this invention is reliable.

[0087] In summary, the above embodiments are merely illustrative examples of preferred embodiments of the present invention and do not encompass all aspects of the invention. Those skilled in the art can make various modifications and refinements without departing from the spirit and scope of the present invention; therefore, the scope of protection of the present invention is defined by the claims.

Claims

1. A method for screening and functional analysis of genes related to improving constipation from multiple traditional Chinese medicine components, characterized in that: The method comprises the following steps: Step one: screening of active ingredients of traditional Chinese medicine compound and corresponding target points of action; Step two: obtaining of target points of constipation disease; Step three: obtaining of intersection target points of traditional Chinese medicine compound target points of action and constipation disease; Step four: construction of active ingredient-target point regulation network of traditional Chinese medicine compound; Step five: construction of PPI protein interaction network; Step six: KEGG and GO enrichment analysis; Step seven: verification of the effect of traditional Chinese medicine compound on improving constipation by using zebrafish model.

2. The method for screening and functionally analyzing genes related to constipation improvement by multi-flavor traditional Chinese medicine ingredients according to claim 1, characterized in that: In the step one, the active ingredients of traditional Chinese medicine compound, i.e. yam, herba rhizomati, angelica sinensis, cistanche deserticola, medlar, astragalus, epimedium and clove, are retrieved and obtained from TCMSP database, and screened according to the criteria of oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18%, and the target points of action corresponding to the screened ingredients are obtained from the database, and finally the target point gene name is standardized by using Uniprot database.

3. The method for screening and functionally analyzing genes related to constipation improvement by multi-flavor traditional Chinese medicine ingredients according to claim 1, characterized in that: In the step two, the target points of constipation disease are collected from GeneCards database, OMIM database, TTD database and PharmGKB database by taking "constipation" as the keyword, and the repeated values are removed.

4. The method for screening and functionally analyzing genes related to constipation improvement by multi-flavor traditional Chinese medicine ingredients according to claim 1, characterized in that: In the step three, the intersection target points are obtained by importing the traditional Chinese medicine compound and the target points of constipation disease into jvenn platform, and a wein diagram is drawn.

5. The method for screening and functional analysis of genes related to constipation improvement by multi-flavor traditional Chinese medicine ingredients according to claim 1, characterized in that: In the step four, the active ingredients and target points of action of traditional Chinese medicine compound are visualized by using Cytoscape 3.7.1 software, and the active ingredient-target point regulation network diagram of traditional Chinese medicine compound is obtained.

6. The method for screening and functionally analyzing genes related to constipation improvement by multi-flavor traditional Chinese medicine ingredients according to claim 1, characterized in that: In the step five, the intersection target points obtained in the step three are imported into STRING12.0 platform to construct the PPI protein interaction network diagram of traditional Chinese medicine compound and constipation.

7. The method for screening and functionally analyzing genes related to constipation improvement by multi-flavor traditional Chinese medicine ingredients according to claim 1, characterized in that: In the step six, the intersection target points of traditional Chinese medicine compound and constipation disease are imported into DAVID database for KEGG and GO enrichment analysis.

8. The method for screening and functionally analyzing genes related to constipation improvement by multi-flavor traditional Chinese medicine ingredients according to claim 1, characterized in that: In the step seven, the effect of traditional Chinese medicine compound on improving constipation is verified by using zebrafish model.

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